Differentiation of Leishmania from pro- to amastigotes involves both qualitative and quantitative morphological and macromolecular changes. To address this problem, we have developed an in vitro culture system for leishmanial growth and differentiation without host cell involvement. We constructed cDNA libraries for both promastigote and amastigote stage of Leishmania donovani. By differential cDNA hybridization, we have isolated three cDNA clones encoding mRNAs expressed at different abundance in these stages. We have designated them as P17, A41 and A45. Nucleotide sequence analysis and comparison of deduced amino acid sequence revealed that the P17 clone is homologous to soybean ribosomal protein S11, A41 is homologous to B. Subtilis spore germination gene (gerC) and A45 has similarity with yeast stress- inducible protein (STI1). The level of RNA for A41 and A45 genes increases several fold when the parasite is grown at 37 degrees C under CO2 atmosphere whereas at the same time P17 mRNA levels decreased by several fold. Southern blot analysis of all the three genes revealed that they belong to a class of single copy genes. Two out of three developmentally regulated genes, i.e. A41 and A45 are present on chromosome 22, whereas P17 gene is present on chromosome 16. The role of differential expression of three genes with respect to Leishmanial development is the subject of future studies.